• Product Name: Smallpox (Vaccinia) Vaccine, Live
• Tradename: ACAM2000
• Manufacturer: Acambis, Inc
• Vaccine Ontology ID: VO_0000003
• CDC CVX code: 75
• Type: Replication competent virus
• Status: Licensed
• Location Licensed: USA (License #1733)
• Host Species for Licensed Use: Human
• Allergen: Neomycin, Polymyxin B
• Preparation: ACAM2000 was prepared from ACAM1000 master seed stock and produced in Vero cells to address the need for rapid large-scale vaccine production (Parrino et al., 2006).
  Specifically, ACAM2000 was manufactured by infecting Vero cells grown on microcarriers under serum-free conditions with the P9 production virus inoculum at an MOI of 0.01–0.2. Virus particles were purified and concentrated. The resulting concentrated bulk vaccine was formulated by dilution with a buffer containing stabilizers to a final potency of 1.0–5.0 × 10^8 pfu/mL, filled into vials containing 0.3 mL (Monath et al., 2004).
• Immunization Route: Percutaneous
• Virulence: It has long been known that vaccinia strains differ with respect to neurovirulence in infant mice. Clones 1, 3, and 5 and the uncloned virus had virulence properties that were unacceptable for consideration as vaccine candidates. The same four viruses that had exhibited excessive virulence in rabbit skin were significantly more neurovirulent than Dryvax1 (p < 0.05, Kaplan—Meier survival distribution, log rank test), whereas clones 2, 4, and 6 were similar to Dryvax1 or less virulent. The more virulent viruses also replicated to higher titer in mouse brain. In these initial experiments Clone 2 did not appear to be attenuated with respect to neurovirulence, but subsequent studies with larger numbers of animals showed significantly higher survival distribution compared to Dryvax1. Clone 2 (renamed ACAM1000) was selected as the candidate for further development, based on its similarity to Dryvax1 in pock formation in rabbit skin but its lower neurovirulence in mice and monkeys. Seed viruses and vaccine produced from each bioreactor run were tested for neurovirulence in suckling mice, using Dryvax1 as a comparator. Plaque-purified vaccinia virus lines were shown to differ significantly in neurovirulence for mice, in their ability to evoke immune responses against the inserted gene product, and in their HindIII restriction maps. The variant viruses often exhibit reduced infectivity and reduced virulence for mice. We found biological and molecular heterogeneity among 6 clones derived from Dryvax1, with some clonal subpopulations (e.g. Clone 3) having dramatically higher virulence and changes at the genomic level. The degree of neurovirulence for suckling mice was used to distinguish vaccine strains with low, moderate, or high pathogenicity (Vilesova et al., 1985). The new vaccine has advantages over first generation vaccines, since it has been produced to modern manufacturing and control standards, is free from adventitious agents , and does not contain subpopulations of virus with undesirable virulence properties (Monath et al., 2004).
  In addition, mice immunized with MVA were protected against lethal infection with a more virulent form of vaccinia virus altered to coexpress IL-4. IL-4 diminishes the cytolytic capacity of CD81 T cells, resulting in delayed viral clearance and increased virulence (Parrino et al., 2006).
• Storage: After reconstitution, ACAM2000 vaccine may be administered within 6 to 8 hours if kept at room temperature (20-25°C, 68-77°F); it should then be discarded as a biohazardous material. Unused, reconstituted ACAM2000 vaccine may be stored in a refrigerator (2-8°C, 36-46°F) up to 30 days, after which it should be discarded as a biohazardous material (FDA: ACAM2000).
• Approved Age for Licensed Use: 1 year of age and older
• Contraindication: Individuals with severe immunodeficiency who are not expected to benefit from the vaccine (FDA: ACAM2000).
• Description: The benefits of cloning appeared to outweigh the recognized risk that a clonal virus population may differ biologically from the ‘genetic swarm’ represented by the animal-skin vaccine. Because it would not be possible to conduct field tests for efficacy, the new vaccine would need to match the licensed vaccine (Dryvax®) as closely as possible in preclinical tests for safety, immunogenicity, and protective activity and in clinical trials for safety and immunogenicity (Monath et al., 2004).
  Clinical trials have been conducted using the NYCBH-derived ACAM2000 vaccinia virus-based vaccine. On the basis of animal studies, ACAM2000 is believed to be less neurovirulent than Dryvax. ACAM2000 was similar to Dryvax in its ability to induce immune responses and in reactogenicity in phase I trials. During phase II and phase III clinical trials, cases of myopericarditis were associated with both ACAM2000 and Dryvax in vaccinia-naive volunteers (Parrino et al., 2006).

• Vaccine Ontology ID: VO_0004094
• Type: Live, attenuated vaccine
• p53 gene engineering:
  • Type: Protein
  • Description: Tumor suppressor protein p53, a nuclear protein, plays an essential role in the regulation of cell cycle, specifically in the transition from G0 to G1. It is found in very low levels in normal cells; however, in a variety of transformed cell lines, it is expressed in high amounts and is believed to contribute to transformation and malignancy. p53 is a DNA-binding protein containing DNA-binding, oligomerization, and transcription activation domains. It is postulated to bind as a tetramer to a p53-binding site and activate expression of downstream genes that inhibit growth and/or invasion and thus function as a tumor suppressor. Mutants of p53 that frequently occur in a number of different human cancers fail to bind the consensus DNA binding site and hence cause the loss of tumor suppressor activity. Alterations of the TP53 gene occur not only as somatic mutations in human malignancies, but also as germline mutations in some cancer-prone families with Li-Fraumeni syndrome (Ober et al., 2002).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000139
  • Description: Complete Freund's adjuvant is used during innoculation, followed by a boost in incomplete Freund's adjuvant (Ober et al., 2002)
• Adjuvant:
  • VO ID: VO_0000142
  • Description: Complete Freund's adjuvant is used during innoculation, followed by a boost in incomplete Freund's adjuvant (Ober et al., 2002)
• Preparation: This vaccine strain was created from the Lister strain by deleting a gene necessary to encode the UDG enzyme, which is essential for a complete cycle of viral replication (Parrino et al., 2006).
• Virulence: The vaccinia virus strain NYVAC was genetically attenuated by deletion of many nonessential genes, including virulence and host range genes, resulting in a strain growing only in primary cells. Passaging in mammalian cells increases virulence in mammals, resulting in new MVA-like strains with unknown safety profiles in humans. The resulting viruses grow exclusively in a complementing permanent cell line, excluding reversion to virulence and obviating the need for primary cells. The WR strain is a vaccinia virus laboratory strain passaged in mouse brain that has unfavorable properties, such as neurovirulence and gonadotropism, not suitable for clinical use. The large deletions characteristic for MVA seemed to suggest that the restriction in host range and virulence was mainly due to these deletions, including the loss of a host range gene and many immune modulatory genes. It will be interesting to see whether an MVA strain first adapted to growth in mammalian cells and then passaged in mouse brain also regains virulence. In contrast to MVA, dVVs with an essential gene deleted cannot regain replication and virulence functions upon passaging in a chosen host. Reversion to virulence can principally be excluded because the vector lacks an essential gene, which restricts its host range to a complementing cell line (Ober et al., 2002).
• Description: dVV-L has been evaluated as a poxvirus vaccine. One great advantage of this approach is that the attenuated virus can be manufactured in a cell line that complements the uracil-DNA-glycosylase (UDG) deficiency, rather than in primary cells or eggs as is often needed for other replication-defective viruses, resulting in an improved safety profile and increased capacity for rapid production (Parrino et al., 2006).

• Vaccine Ontology ID: VO_0004095
• Type: Subunit vaccine
• A33R from Vaccinia virus (strain: WR (Western Reserve)) gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-A33R; associates with A36R; involved in CEV-cell adherence and actin tail formation (NCBI).
  • Detailed Gene Information: Click Here.
• A34R gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-A34R; involved in CEV cell adherence and actin tail formation (NCBI).
  • Detailed Gene Information: Click Here.
• A36R gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-A36R; interacts with A33R and used in actin tail formation (NCBI).
  • Detailed Gene Information: Click Here.
• B5R from Vaccinia virus (strain: WR (Western Reserve)) gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-B5R; required for trans-Golgi/endosomal membrane-wrapping of IMV (NCBI).
  • Detailed Gene Information: Click Here.
• D8L gene engineering:
  • Type: Protein
  • Description: Infectious intracellular mature virions (IMV), containing a complex core structure and an outer membrane with nonglycosylated viral proteins, are assembled in factory regions within the cytoplasm of vaccinia virus-infected cells. Some IMV migrate out of the factories, become wrapped with an additional double membrane containing viral glycoproteins, and are then transported on microtubules to the periphery of the cell. The outer of the two added membranes fuses with the plasma membrane during exocytosis, and the resulting extracellular particles consist of an IMV surrounded by one extra fragile membrane (Fogg et al., 2004).
  • Detailed Gene Information: Click Here.
• H3L gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-H3L; involved in IMV maturation (NCBI).
  • Detailed Gene Information: Click Here.
• L1R from Vaccinia virus (strain: WR (Western Reserve)) gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-L1R; target of neutralizing antibody; S-S bond formation pathw thiol substrate; myristylprotein (NCBI).
  • Detailed Gene Information: Click Here.
• VACVgp196 gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• VACVgp200 gene engineering:
  • Type: Protein
  • Description: The cell-associated and released extracellular virions (EV) are thought to be largely responsible for direct cell-to-cell and long-range virus spread within a host, respectively (Fogg et al., 2004).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0001238
  • Description: Either a Ribi adjuvant system consisting of MPL+TDM or the saponin adjuvant QS-21 was used (Fogg et al., 2004)
• Adjuvant:
  • VO ID: VO_0001310
• Preparation: Soluble forms of L1, A33, and B5 were purified. Recombinant proteins (10 µg) were diluted in PBS with the adjuvant for a total injection volume of 0.1 ml. Monophosphoryl-lipid A plus trehalose dicorynomycolate emulsion (MPL+TDM) was prepared immediately before each immunization according to manufacturer's instructions (Fogg et al., 2004).
• Virulence: (Fogg et al., 2004)
• Description: Both intracellular mature virus (IMV) and EV are infectious, but they contain different viral outer membrane proteins, bind to cells differently and have different requirements for entry. Although the entry process is not well understood, a model consistent with available data is that IMV fuse directly with plasma membrane, whereas EV entry involves endocytosis, low-pH-induced disruption of the outer membrane, and fusion of the exposed IMV with the endosomal membrane. Protein subunit vaccines have been evaluated in mice (Fogg et al., 2004).
  Recombinant proteins of the outer membranes of IMV and EEV forms of vaccinia virus were used individually or in combination to immunize mice before i.n. challenge with a lethal dose of the WR strain of vaccinia virus. Vaccination with the individual proteins afforded partial protection; complete protection was achieved with 3 doses of the 3-protein IMV–EEV combination vaccine (Parrino et al., 2006).

• Vaccine Ontology ID: VO_0004150
• Type: Inactivated or "killed" vaccine
• Antigen: Two strains of vaccinia virus (VV) were used for antigens: VV Western Reserve strain (VVWR) and recombinant Western Reserve strain (VVWR-Luc). The recombinant strain is the same as the VVWR except for expression of firefly luciferase from the pH 7.5 early/late promoter (Bielinska et al., 2008).
• Adjuvant:
  • VO ID: VO_0001319
• Preparation: The viruses killed by incubation were incubated for 3 h at 37°C in 10% NE. Nasal instillation killed virus was diluted to obtain either 1 x 10^3 PFU or 1 x 10^5 PFU per dose in 1% NE. Vaccine formulations containing formalin-killed virus (Fk) were prepared by incubation of VV (about 10^8 PFU/ml) with 0.1% formalin at room temperature for 3 h. This mixture was then diluted in either saline or 1% NE to 1 x 10^3 or 1 x 10^5 PFU per dose to reduce the formalin to nontoxic concentrations for intranasal (i.n.) immunization (Bielinska et al., 2008).

• Vaccine Ontology ID: VO_0004091
• Type: Attenuated Lister strain
• A33R from Vaccinia virus (strain: WR (Western Reserve)) gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-A33R; associates with A36R; involved in CEV-cell adherence and actin tail formation (NCBI).
  • Detailed Gene Information: Click Here.
• A34R gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-A34R; involved in CEV cell adherence and actin tail formation (NCBI).
  • Detailed Gene Information: Click Here.
• A36R gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-A36R; interacts with A33R and used in actin tail formation (NCBI).
  • Detailed Gene Information: Click Here.
• A56R gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-A56R; EEV; type-I membrane glycoprotein; inhibits cell fusion (NCBI).
  • Detailed Gene Information: Click Here.
• A21L gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• B5R from Vaccinia virus (strain: WR (Western Reserve)) gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-B5R; required for trans-Golgi/endosomal membrane-wrapping of IMV (NCBI).
  • Detailed Gene Information: Click Here.
• F13L gene engineering:
  • Type: Protein
  • Description: Similar to VACCP-F13L; phospholipase motif, required for IEV formation (NCBI).
  • Detailed Gene Information: Click Here.
• Preparation: Virus suspensions were diluted serially in phosphate-buffered saline (PBS) supplemented with adjuvant and overlaid with a suspension of 1% carboxyl methyl cellulose in DMEM supplemented to contain 5% FBS (Empig et al., 2006).
• Virulence: RPV is a virulent strain of vaccinia virus that produces high levels of detectable EEV. In infected rabbits, it causes a generalized, disseminated infection, resulting in death in the majority of cases. However, LC16m8, despite the B5R mutation, is equivalent to Dryvax in the capacity to protect rabbits from lethal RPV challenge (Empig et al., 2006).
• Description: LC16m8 was developed and widely used in Japan prior to the global eradication of smallpox. Numerous preclinical safety studies in several animal species were conducted comparing LC16m8 to the parental strain Lister, each showing that the attenuated vaccinia virus strain, in contrast to Lister, was incapable of invading the central nervous system (CNS). LC16m8 also was not neuroinvasive in cortisone-treated immunocompromised mice. Clinical trials in Japan, in which over 10,000 children received LC16m8, demonstrated enhanced safety of this attenuated vaccine in comparison to Lister, while confirming that its immunogenicity was unaltered (Empig et al., 2006).

• Vaccine Ontology ID: VO_0004092
• Type: Replication-defective virus
• Preparation: A vial of MVA passage 572 was plaque-purified, propagated in chick embryo fibroblasts, and purified by sedimentation through a sucrose cushion (Earl et al., 2004).
• Virulence: (Earl et al., 2004; Meseda et al., 2005)
• Description: Modified vaccinia Ankara (MVA) has been studied most extensively out of the replication-defective vaccines. MVA has an excellent safety profile and could be used in groups in whom Dryvax is currently contraindicated. MVA was given to 120,000 people in Germany in the 1970s, followed by vaccination with live virus Elstree. MVA was safe but was not field-tested because smallpox was not present in Europe at that time. MVA has since been evaluated in animal models and in human studies. In phase I human clinical trials, MVA was found to be safe and immunogenic on its own and found to prime for greater immune responses and attenuate lesion formation if given in advance of Dryvax vaccination. MVA is also being evaluated in persons with contraindications to live virus vaccine such as atopic dermatitis and immunosuppression (Parrino et al., 2006).

• Vaccine Ontology ID: VO_0004093
• Type: Replication defective virus
• Preparation: NYVAC was derived from the Copenhagen strain and developed by selective deletion of 18 open reading frames (ORFs) (Parrino et al., 2006).
• Virulence: (Belyakov et al., 2003; Edghill-Smith et al., 2003)
• Description: Smallpox vaccination induced significantly larger skin lesions in immunocompromised macaques than in healthy macaques. Vaccination of immunocompromised macaques with the genetically-engineered, replication-deficient poxvirus NYVAC, before or after retrovirus infection, was safe and lessened the severity of Dryvax-induced skin lesions. Neutralizing antibodies to vaccinia were induced by NYVAC, even in macaques with severe CD4+ T cell depletion, and their titers inversely correlated with the time to complete resolution of the skin lesions. Together, these results provide the proof of concept, in macaque models that mirror human immunodeficiency virus (HIV) type 1 infection, that a prime-boost approach with a highly attenuated poxvirus followed by Dryvax increases the safety of smallpox vaccination, and they highlight the importance of neutralizing antibodies in protection against virulent poxvirus (Edghill-Smith et al., 2003).

• Vaccine Ontology ID: VO_0004149
• Type: Subunit vaccine
• Antigen: This vaccine uses the A27L and D8L proteins from the intracellular mature virus form and the B5R protein from the extracellular enveloped virus form of the vaccinia virus (Berhanu et al., 2008).
• Adjuvant:
  • VO ID: VO_0001250
  • Description: The adjuvant used in this vaccine was Monophosphoryl Lipid A and Trehalose Dicorynomycolate (MPL-TDM) (Berhanu et al., 2008).
• Preparation: Vaccines contained 10 micrograms of A27L, D8L, and B5R in 100 microliters of phosphate buffered saline mixed with 100 microliters of MPL-TDM (Berhanu et al., 2008).

• Vaccine Ontology ID: VO_0004096
• Type: DNA
• A27L gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• A33R from Monkeypox virus (strain: Zaire-96-I-16) gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• B5R from Monkeypox virus (strain: Zaire-96-I-16) gene engineering:
  • Type: Protein
  • Description: (Hooper et al., 2004)
  • Detailed Gene Information: Click Here.
• L1R from Monkeypox virus Zaire-96-I-16 gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• Preparation: The 4pox DNA vaccine contained two IMV-specific genes (L1R and A27L) and two EEV-specific genes (A33R and B5R) (Hooper et al., 2004).
• Virulence: (Hooper et al., 2004)
• Description: DNA vaccine strategies have been investigated in animal models. A DNA vaccine composed of 4 vaccinia virus genes protected rhesus macaques from severe disease, with the animals exhibiting mild clinical and laboratory abnormalities, after challenge with a lethal dose of monkeypox virus. When vaccinated with a single gene (L1R), macaques developed severe, but not fatal, disease. Heterologous prime-boost strategies have also been evaluated. Priming BALB/c mice with DNA vaccine resulted in greater immune responses after boosting with live vaccinia virus compared with controls (Parrino et al., 2006).

Human Response

• Host Strain: Healthy adults aged 18–29 years.
• Vaccination Protocol: Clinical development of ACAM2000 commenced with a Phase 1 open-label trial in 100 healthy adults without prior smallpox vaccination. The primary endpoint was the proportion of subjects with a major cutaneous reaction assessed at any time-point from Day 7 (±2) through Day 15 (±2). Fifty-six percent of subjects were male. The majority (89%) were Caucasian; the remaining subjects were African-American (7%), Asian (3%), or Hispanic (1%). The mean age was 23 years, with a range of 18 to 29 years (Monath et al., 2004).
• Persistence: Of the 99 subjects who experienced a major cutaneous reaction, 9% had a major cutaneous reaction by Day 3, and the rest experienced a major cutaneous reaction by Day 7. The progression of the cutaneous reaction and its size and appearance were similar to those observed in the trials of ACAM1000. The great majority (96%) developed ≥four fold increases in neutralizing antibodies. The geometric mean neutralizing antibody titer on Day 30 was 225. Four (4%) of 100 subjects did not have a four-fold increase in neutralizing antibody titer on Day 30. However, these 4 subjects all had a major cutaneous reaction by Day 7 (Monath et al., 2004)
• Side Effects: With the diminishing threat of smallpox and increased focus on adverse events, vaccination in the United States was discontinued in 1972 for the general public and in 1989 for military personnel. The safety of ACAM2000 was assessed by documentation of adverse events, physical examination findings, lymph node assessments, measurements of vital signs, and clinical laboratory tests, including hematology, clinical chemistry, and urinalysis. Subjects in the study kept a diary of adverse events and took daily oral temperatures. There were no serious adverse events. All 70 subjects (100%) experienced at least one treatment-emergent, expected adverse event during the study. The adverse events were generally mild and did not interfere with the subjects’ daily activities. One subject experienced a serious adverse event, a single new onset seizure on Day 8; this event was considered by the investigator to be remotely related to the study vaccine. The most commonly reported treatment-emergent adverse events were related to the vaccination site and associated lymphadenitis, and the majority of adverse events reported were assessed as mild or moderate in intensity. Elevated temperature was reported as an adverse event for 9 (9%) subjects. Fortunately, cardiac adverse events appear to be self-limiting (Monath et al., 2004).
• Efficacy: Ninety-nine percent of the subjects experienced a successful vaccination (Monath et al., 2004).
• Description: Phase 1 clinical trials of ACAM1000 and 2000 indicate that the original goal of producing a second generation vaccine that closely matched the safety and immunogenicity of calf-skin vaccine (Dryvax®) was met. The cutaneous, antibody, and T cell responses in primary vaccinees were similar to those elicited by Dryvax®. The appearance and size of the cutaneous lesion and pattern of virus shedding from the vaccination site were also similar. Phase 2 trials in naïve and previously vaccinated subjects have been completed to define the dose response, and to extend safety and immunogenicity data. Phase 3 clinical trials are in progress (Monath et al., 2004).

Human Response

• Host Strain: rhesus macaque (Macaca mulata)
• Vaccination Protocol: The challenge experiment included 4 groups: group 1 consisted of 3 monkeys vaccinated with the 4pox DNA vaccine, group 2 consisted of 2 monkeys vaccinated with the L1R DNA vaccine, group 3 (negative controls) consisted of 3 monkeys vaccinated with a Hantaan virus DNA vaccine, and group 4 (positive controls) consisted of 2 monkeys vaccinated with the human smallpox vaccine (Dryvax). The L1R DNA vaccine was tested to determine the degree to which vaccination with a single immunogen eliciting IMV-neutralizing antibodies could confer protection. The DNA vaccines were administered by gene gun. Five weeks before challenge, all monkeys, except the monkeys vaccinated with Dryvax and one of the negative controls, were vaccinated with new preparations of the same DNA vaccine they had received 1-2 years earlier. This booster vaccination was administered to affirm that immunological memory had been elicited by the initial vaccination series and to ensure robust responses to the DNA vaccines with the intent to prove concepts rather than explore minimal requirements for protection. Based on the dosing experiments, a dose of 2 x 10^7 PFU was chosen for the vaccine evaluation experiment. Vaccinated monkeys were challenged with MPOV-Z79 by i.v. injection into the right or left saphenous vein. At 2-day intervals, whole-blood, serum, and throat swab samples were collected, and rectal temperature, pulse, and blood oxygen saturation were measured (Hooper et al., 2004).
• Persistence: Gene gun vaccination with the 4pox DNA vaccine or the L1R DNA vaccine elicited a memory response that was maintained for at least a year and up to 2 years (Hooper et al., 2004).
• Side Effects: Although VACV is highly immunogenic and is known to confer long-lasting protective immunity to smallpox, the adverse events associated with the present smallpox vaccine (i.e., Dryvax) pose a significant obstacle to successful vaccination campaigns. Adverse events historically associated
  with VACV range from the nonserious (e.g., fever, rash, headache, pain, and fatigue) to life threatening (e.g., eczema vaccinatum, encephalitis, and progressive vaccinia). Serious adverse events that are not necessarily causally associated with vaccination, including myocarditis and/or myopericarditis, have been reported during past and present smallpox vaccination programs. Several adverse cardiac events reported in the first 4 months of the 2003 civilian and military vaccination campaigns prompted the CDC to revise their recommendations for exclusion of potential smallpox recipients to include those persons with heart disease or several other conditions. In addition, identifying protective immunogens might allow the development of a subunit smallpox vaccine that affords protection with negligible adverse events (Hooper et al., 2004).
• Efficacy: Monkeys vaccinated with the 4pox DNA vaccine were protected not only from lethal monkeypox but also from severe disease. This is the first demonstration that vaccination with a combination of VACV immunogens, rather than the whole infectious virus, is sufficient to protect NHPs against any poxvirus disease (Hooper et al., 2004).
• Description: Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine administered to the skin, the vaccine itself can pose a serious health risk. The present study demonstrates that monkeys vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R), which encodes a target of neutralizing antibodies, developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible (Hooper et al., 2004).

Mouse Response

• Host Strain: 3-4 day-old outbred ICR mice
• Vaccination Protocol: Groups of mice were inoculated with graded doses (0.3 to 3.0 log10 pfu) (Monath et al., 2004).
• Persistence: Survival analysis showed that ACAM1000 and ACAM2000 did not differ from one another but had significantly longer survival than Dryvax (Monath et al., 2004).
• Side Effects: We showed that ACAM1000 and ACAM2000 were significantly less neurovirulent for mice and monkeys than the parental Dryvax1 virus, presumably. ACAM2000 should be less likely to cause post-vaccinal encephalitis in humans. However, the pathogenesis of postvaccinal
  encephalitis is still uncertain. Vaccinia virus has been isolated from CSF and brain, suggesting that the virus invades the central nervous system in humans (Monath et al., 2004).
• Efficacy: The median lethal dose (LD50) and 90% lethal dose (LD90) were higher for mice receiving ACAM2000 and ACAM1000 compared to Dryvax (Monath et al., 2004).
• Description: The neurovirulence profiles of ACAM2000 and ACAM1000 vaccines were compared to Dryvax in a lethal dose assay (Monath et al., 2004).

Mouse Response

• Host Strain: Young adult BALB/c mice.
• Vaccination Protocol: Groups of 5 mice were immunized with graded doses (4 to 7 log10 PFU/mL) of ACAM 2000 and then challenged 3 weeks later with 100 LD50 of vaccinia WR virus. Survival and body weight were recorded daily for 14 days after challenge (Monath et al., 2004).
• Persistence: The survival times were not statistically different between treatment groups (Monath et al., 2004).
• Side Effects: We showed that ACAM1000 and ACAM2000 were significantly less neurovirulent for mice and monkeys than the parental Dryvax1 virus, presumably. ACAM2000 should be less likely to cause post-vaccinal encephalitis in humans. However, the pathogenesis of postvaccinal
  encephalitis is still uncertain. Vaccinia virus has been isolated from CSF and brain, suggesting that the virus invades the central nervous system in humans (Monath et al., 2004).
• Efficacy: Protective efficacy of the 3 viruses tested was similar (Monath et al., 2004).
• Description: Mice were used to compare the protective efficacy of immunization with ACAM2000, ACAM1000, and Dryvax (Monath et al., 2004).

Mouse Response

• Host Strain: Mice (BALB/c/SCID, 6 to 8 weeks old).
• Vaccination Protocol: Groups of four immunodeficient mice were challenged subcutaneously (s.c.) with high doses of the wild-type Lister strain or the nonreplicating Lister- or MVA-based vectors (Ober et al., 2002).
• Persistence: Using the wild-type Lister virus, doses of ≥10^6 PFU led to a progressive vaccinia virus infection within a 2-month observation period (Ober et al., 2002).
• Side Effects: The defective Lister strain-based viruses did not induce any signs of progressive disease. The nonreplicating virus was tolerated without any visible signs of discomfort of the mice at doses of 10^7 and 10^8 PFU. The highest dose of 10^9 PFU was accompanied by mild signs of sickness in the first few days, which disappeared later. At 4 weeks after challenge a lesion was observed at the injection site, which subsequently healed. In summary, not only in the in vitro system but also in vivo in immunodeficient animals, dVVs based on the Lister strain are as well tolerated as the MVA-based viruses (Ober et al., 2002).
• Efficacy: A more suitable smallpox prevaccine for immunocompromised subjects, dVV was highly protective in a preclinical challenge model, induced antibodies and CTLs similarly to MVA, and was as safe as MVA-based recombinants in immunodeficient mice. In addition, reversion to virulence can principally be excluded because the vector lacks an essential gene, which restricts its host range to a complementing cell line (Ober et al., 2002).
• Description: The main concern about the use of replication-competent viruses in immunotherapy is that severe adverse effects may occur in immunocompromised patients. The mouse model was used to more thoroughly address the safety question in an in vivo model (Ober et al., 2002).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Female 5- to 6-week-old BALB/c mice were purchased from Taconic (Germantown, N.Y.). 15 µg of QS-21 aliquots (2 mg/ml in water) were used for each immunization. Proteins were administered at 3-week intervals. Blood was collected from the tail vein 1 day prior to each immunization.
  One day prior to challenge, serum samples were collected and mice were weighed. On the day of challenge, an aliquot of purified VV-WR was thawed, sonicated, and diluted in PBS. Mice were anesthetized by inhalation of isoflurane and inoculated i.n. with a 20-µl suspension of 1 x 10^6 or 2 x10^7 PFU of VV-WR. Mice were weighed daily for 2 w following challenge and were euthanatized when they lost 30% of their initial body weight (Fogg et al., 2004).
• Persistence: (Fogg et al., 2004)
• Side Effects: (Fogg et al., 2004)
• Efficacy: Complete survival was obtained with the combination of all three proteins. Although there is a need for safer vaccines, it is difficult to evaluate their efficacy in the absence of human smallpox or information regarding the correlates of immunity (Fogg et al., 2004).
• Description: Soluble forms of several vaccinia virus IMV and EV membrane proteins have been engineered to learn more about immunity to poxviruses and to test the proteins as components of a vaccine. The present study involves recombinant L1, A33, and B5 proteins individually or in combinations and then challenged the mice (Fogg et al., 2004).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Mice were vaccinated with 10 to 15 µl of vaccine formulation per naris by use of a pipette tip. Emulsion was applied slowly to minimize bronchial distribution and swallowing of the material (Bielinska et al., 2008).
• Immune Response: The intranasal vaccination resulted in both systemic and mucosal anti-VV immunity, virus-neutralizing antibodies, and Th1-biased cellular responses (Bielinska et al., 2008).
• Side Effects: Mice protected with VV/NE immunization did have clinical symptoms more extensive than animals vaccinated by scarification post-challenge (Bielinska et al., 2008).
• Challenge Protocol: Aliquots of purified recombinant VVWR-Luc or VVWR were thawed and diluted in saline on the day of the challenge. Mice were challenged i.n. with a 20-µl suspension of 2 x 10^6 PFU live VVWR-Luc, corresponding to 10 times the 50% lethal dose, or with live VVWR doses ranging from 1 x 10^7 to 3.2 x 10^3 in fivefold dilutions (Bielinska et al., 2008).
• Efficacy: Nasal vaccination with VV/NE vaccine produced protection against lethal infection equal to vaccination by scarification, with 100% survival after challenge (Bielinska et al., 2008).

Mouse Response

• Host Strain: A/NCR
• Vaccination Protocol: Three groups of 30 4–6 week old A/NCR mice each were vaccinated at the base of the tail with LC16m8 (approximately 2 × 10^5 PFU). Forty-one days post-vaccination, sera were collected for assessment of virus-specific antibody responses prior to challenge. Forty-nine days post-vaccination, animals were challenged with ECTV delivered by aerosol. ECTV was suspended in DMEM without FBS and inoculated by using a nose-only inhalation exposure system. The remaining mice were observed for 21 days for signs of disease and mortality. Animals were evaluated daily for weight assessment and clinical symptoms (Empig et al., 2006).
• Persistence: (Empig et al., 2006)
• Side Effects: Despite limiting vaccination to healthy individuals in recent vaccination campaigns, adverse reactions were still observed, highlighting the need for a safer yet equally protective alternative to Dryvax. However, no serious adverse reactions, such as encephalitis, were observed during the early use of LC16m8 vaccine (Empig et al., 2006).
• Efficacy: Mice immunized with LC16m8 were protected against lethal ECTV infection. LC16m8 generated antibody responses in mice that exceeded those generated by Dryvax (Empig et al., 2006).
• Description: To evaluate the protective efficacy of LC16m8 in comparison to Dryvax, the study employed both rabbit and mouse models of poxvirus disease (Empig et al., 2006).

Mouse Response

• Host Strain: BALB/cByJ
• Vaccination Protocol: To compare the effectiveness of various routes of MVA immunization, male BALB/cByJ mice (obtained from the Jackson Laboratory, Bar Arbor, ME) were immunized through 3 different routes at doses from 10^6 to 10^8 pfu, and sera were collected every 3 weeks for 15 weeks for evaluation of Dryvax-specific antibody by ELISA using inactivated virus (Meseda et al., 2005).
• Persistence: The antibody response to vaccination was observed in mice over a relatively long period of time (12–15 weeks) following the initial dose of vaccine. By each measurement, the elicited immune response was stable over this time frame for both Dryvax and MVA. Further, when animals received a second dose of MVA, the antibody response was elevated compared to a single immunization, and was stable for the remainder of the observation period (6 to 9 weeks) (Meseda et al., 2005).
• Side Effects: A safer smallpox vaccine could benefit the millions of people that are advised not to take the current one because they or their contacts have increased susceptibility to severe vaccine side effects. Because the correlates of smallpox protection are unknown, findings of similar humoral and cellular immune responses to MVA and Dryvax in NHPs and substantial protection against a severe monkeypox virus challenge are important steps in the evaluation of MVA as a replacement vaccine for those with increased risk of severe side effects from the standard live vaccine, or as a pre-vaccine. As a result of extreme attenuation, MVA causes no adverse effects even when high doses are injected into immunedeficient NHPs. No adverse local or systemic effects were noted after vaccination with MVA. As expected, pustular skin lesions did develop after Dryvax (Earl et al., 2004).
  Significant adverse events are associated with vaccination with the currently licensed smallpox vaccine. Candidate new-generation smallpox vaccines, such as MVA, produce very few adverse events in experimental animals and in limited human clinical trials conducted near the end of the smallpox eradication campaign. MVA was administered to more than 120,000 individuals in the latter stages of the smallpox eradication campaign without significant adverse events, although the thoroughness of safety data monitoring at that time is unclear. In addition to a vaccination strategy that employs multiple immunizations of MVA, alternative smallpox vaccination strategies may include an initial vaccination with non-replicating virus vaccine followed by a second immunization with a traditional replicating virus vaccine in order to reduce the possibility of vaccine-associate adverse events due to replicating vaccinia virus. Such a scheme of vaccination may be considered as a means of reducing the rate of adverse events associated with traditional smallpox vaccination, provided that vaccine efficacy is not compromised (Meseda et al., 2005).
• Efficacy: Mice immunized intradermally (i.d.) with either 10^8 pfu of MVA, or a prime-boost combination of 10^8 pfu of MVA followed by either 10^6 pfu of Dryvax or 10^8 pfu of MVA survived an intranasal (i.n.) challenge with 25 LD50s of vaccinia virus WR. Furthermore, vaccination with a single dose of 10^8 pfu of MVA resulted in a minimal weight loss (<10%), as did a vaccination combination of 10^8 pfu of MVA followed by 10^6 pfu of Dryvax. When mice that were immunized with a lower dose of 10^6 pfu of MVA were challenged with i.n. vaccinia virus WR, 4/5 survived a challenge with 10 LD50s at either 6 weeks or 12 weeks post-vaccination. When mice received a single immunization of 10^6 pfu of MVA and were challenged with 25 LD50s, 4/5 survived challenge at 6 weeks post-vaccination and 3/5 survived challenge at 12 weeks post-vaccination. In contrast, all animals receiving 10^6 MVA and boosted 6 weeks later with either 10^6 pfu of MVA or 10^6 pfu of Dryvax survived. These results indicate that combinations of MVA are as effective as Dryvax in eliciting immune responses and inducing protective immunity in a mouse model (Meseda et al., 2005).
• Description: The aim of the present study was to compare the immunogenicity and protective ability of MVA (a leading candidate new-generation smallpox vaccine) to the licensed smallpox vaccine Dryvax in a mouse model of vaccination. MVA is a replication-defective vaccinia virus derived from the Ankara strain by more than 500 passages through primary chicken embryo fibroblasts (CEF). This virus grows to high titer in CEF cells but replicates poorly, if at all, in human cells (Meseda et al., 2005).

Mouse Response

• Host Strain: BALB/c, B cell-deficient, and CD1 KO–/– mice
• Vaccination Protocol: Female BALB/c mice (6–10 weeks old, purchased from Frederick Cancer Research Facility, Frederick, MD), B cell-deficient (Taconic Farms), and CD1 KO-/- (CD1KO, from M. Grusby) mice were innoculated with NYVAC at doses from 10^3 to 10^7 pfu. For comparison, and as a positive control, immunization with Wyeth human vaccine strain of vaccinia virus was given by tail scratch (corresponding to skin scratch used for human vaccination). One month after immunization, mice were challenged with 10^6 pfu of WR by intranasal (i.n.) inoculation (Belyakov et al., 2003).
• Persistence: (Belyakov et al., 2003)
• Side Effects: None were mentioned. Replication-defective strains might be valuable as a preliminary immunization to reduce the risk of serious adverse ffects of conventional smallpox vaccination (Belyakov et al., 2003).
• Efficacy: Protection at most doses of NYVAC given i.m. was roughly comparable to that produced by the corresponding doses of MVA given i.m., and no statistically significant difference was detected. It was found that i.m. injection with MVA induced protection of immunized animals in a dose-dependent manner. A dose of 10^7 pfu of MVA given i.m. induced complete protection against challenge with WR (Belyakov et al., 2003).
• Description: At sufficient doses, the protection provided by modified NYVAC replication-deficient vaccinia viruses, safe in immunocompromised animals, was equivalent to that of the licensed Wyeth vaccine strain against a pathogenic vaccinia virus i.n. challenge of mice. A similar variety and pattern of immune responses were involved in protection induced by modified vaccinia Ankara and Wyeth viruses. For both, antibody was essential to protect against disease, whereas neither effector CD4+ nor CD8+ T cells were necessary or sufficient. However, in the absence of antibody, T cells were necessary and sufficient for survival and recovery. Also, T cells played a greater role in control of sublethal infection in unimmunized animals. These properties, shared with the existing smallpox vaccine, provide a basis for further evaluation of these replication-deficient vaccinia viruses as safer vaccines against smallpox or against complications from vaccinia virus (Belyakov et al., 2003).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Six-week old mice were immunized three times subcutaneously at weeks 0, 3, and 5 (Berhanu et al., 2008).
• Immune Response: Three immunization with the proteins alone induced potent neutralizing antibody reponses (Berhanu et al., 2008).
• Challenge Protocol: Five weeks or two weeks after the last immunization, mice were challenged intranasally with with 20 LD50 VV-WR in 20 microliters of PBS by applying equally between the two nares (Berhanu et al., 2008).
• Efficacy: Immunization provided complete protection against lethal viral challenge. Several linear B-cell epitopes within the three proteins were recognized by serum from the immunized mice. In addition protein-specific cellular responses were detected in spleens of immunized mice by gamma interferon (Berhanu et al., 2008).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Adult (16–23 g) female BALB/c mice were vaccinated in the skin of the thigh using an Easy Vax™ DNA vaccine delivery system to deliver the vaccine plasmids on weeks 0, 3, and 8. Anesthetized mice were scarified by placing 10 μl of PBS containing live VACV on the tail. A 26 gauge 5/8" needle was used to scratch the tail to facilitate infection/vaccination. A lesion (pock) at the site of scarification on d 10 indicated successful vaccination. Mice were anesthetized and weighed before i.n. injection of 50 μl of PBS containing 2 × 10^6 pfu of VACV strain IHD-J using a plastic pipette tip. After challenge, mice were observed and weighed daily for 3 weeks (Hooper et al., 2006).
• Persistence: (Hooper et al., 2006)
• Side Effects: There are several drawbacks to the current anthrax vaccines including nonserious and serious adverse events that make the vaccine contraindicated in large segments of the population (e.g., persons who are immunodeficient, immunosuppressed, pregnant, breastfeeding, or have history of cardiac disease), and because this vaccine results in a localized skin infection containing infectious virus (i.e. pock), the infection can spread to other sites on the body (e.g. ocular autoinoculation) or to persons who come in close contact with the vaccinee. Identification of the genes associated with protective immunity and, conversely, the genes associated with adverse events unrelated to dissemination or transmission will be important for characterizing the next-generation smallpox vaccines and for engineering future smallpox vaccines (Hooper et al., 2006).
• Efficacy: Mice vaccinated with the 4pox DNA vaccine using the Easy Vax™ device were completely protected from i.n. challenge with >10 LD50 of VACV, strain IHD-J (Hooper et al., 2006).
• Description: The enhanced immunogenicity of DNA vaccines delivered by gene gun likely involves the direct introduction of plasmid DNA to cells in the skin, including specialized antigen-presenting cells (APCs). While the gene gun has yielded among the most promising immune responses for a DNA vaccine thus far, there is the possibly that all of the criteria required for successful product development will not be satisfied. Hence, it is important to continue to evaluate alternative technologies that might better facilitate the development of licensed human vaccines. Alternative means of delivering DNA vaccines under investigation include the use of electric field technologies. Electroporation is a process whereby cells are transiently permeabilized by high-intensity electric field pulses. The present study tests a novel device capable of targeting electroporation to the dermis using a microneedle array. The plasmid DNA is dried onto the tips of the microneedles, which are inserted into the skin where the DNA dissolves in interstial fluid and is then transfected into the surrounding cells by electroporation (Hooper et al., 2006).

Monkey Response

• Host Strain: Cynomolgous macaque (Macaca fascicularis)
• Vaccination Protocol: Monkeys were inoculated with 10^8 plaque-forming units (PFU). 24 monkeys were divided into 4 groups: group 1 received an inoculation with 10^8 PFU of MVA at t = 0 and a second 2 months later; group 2 received one injection with 10^8 PFU of MVA followed 2 months later by a standard percutaneous inoculation with Dryvax; group 3 received nothing at t = 0 and 1 Dryvax inoculation 2 months later; group 4 served as the unimmunized control (Earl et al., 2004).
• Persistence: The response to the first MVA inoculation was detected at 1 week, peaked at 2-4 weeks, and was boosted 1 week after the second MVA dose (Earl et al., 2004).
• Side Effects: MVA caused no adverse effects, even when high doses were injected into immune-deficient NHPs (Earl et al., 2004).
• Efficacy: All immunized animals remained clinically well (Earl et al., 2004).
• Description: As vaccines can no longer be tested for their ability to prevent smallpox, licensing will necessarily include comparative immunogenicity and protection studies in non-human primates (NHPs). Here, a highly attenuated MVA is compared with the licensed Dryvax vaccine in an NHP model (Earl et al., 2004).

Monkey Response

• Host Strain: Indian rhesus macaques
• Vaccination Protocol: Twenty-five monkeys were enrolled: 6 of the macaques were immunocompetent (groups 1 and 2), and macaques in group 1 were vaccinia naive. Macaques in group 2 had been exposed previously to the attenuated nef- SIVmac239 strain. They were immunized with a single inoculation of NYVAC 1 month before Dryvax vaccination. Group 3 included 7 macaques, 3 that had been infected with the chimeric SHIV 89.6 PD strain for 8 months, 3 that had been infected with the SIVmac251 strain for 12 months, and 1 that had been infected with the nef- SIVmac239 strain for 32 months. Four macaques (group 5) that, at first, had been infected with the same SIVmac251 strain and that subsequently were vaccinated with 3 inoculations of NYVAC, at weeks 10, 19, and 23 after infection (for macaques 480, 644, and H684) or at weeks 42, 48, and 54 after infection (for macaque 3143), were used. The overall time of SIVmac251 infection was 41 months for macaques 480 and 644 and 25 months for macaques H684 and 3143. Four macaques had been infected with SHIV 89.6 PD for 12 months. They were vaccinated with 3 inoculations of NYVAC (10^8 pfu) 6 weeks apart and were challenged with Dryvax 6 months after the final NYVAC immunization. All 25 macaques were vaccinated with Dryvax at the same dose at the times indicated. In brief, the bifurcated needle was immersed in the vaccine suspension and was used to poke the skin 15 consecutive times, in accordance with US Food and Drug Administration (FDA) guidelines. The lesions that developed after smallpox vaccination were photographed every 2 days and were imaged by manually defining the topographic contours of the affected skin (Edghill-Smith et al., 2003).
• Persistence: The immunocompromised macaques were vaccinated with NYVAC at 6 months to a maximum of 36 months before Dryvax challenge, suggesting that this vaccine is able to induce lasting immune responses even as CD4+ helper T cells are progressively depleted. However, the lag between NYVAC and Dryvax vaccinations appears to be important (Edghill-Smith et al., 2003).
• Side Effects: The prime-boost approach with a highly attenuated poxvirus followed by Dryvax increases the safety of smallpox vaccination (Edghill-Smith et al., 2003).
• Efficacy: The prime-boost approach with a highly attenuated poxvirus followed by Dryvax increases the safety of smallpox vaccination, and highlights the importance of neutralizing antibodies in protection against virulent poxvirus (Edghill-Smith et al., 2003).
• Description: The replication competence of live vaccines, such as the only currently available smallpox vaccine, Dryvax, may pose safety concerns when injected in individuals with congenital, acquired, or iatrogenic immunodeficiency. Because the number of patients with immunodeficiency has increased worldwide as a result of the HIV-1 epidemic, the increase in the number of organ transplants, and aggressive chemotherapy in patients with cancer, the risks associated with Dryvax vaccination may affect a larger portion of the population than before. It has been hypothesized that immunization of immunocompromised individuals, with highly attenuated poxviruses, may ameliorate the clinical outcome of Dryvax vaccination. In macaques with modest to severe depletion of CD4+ T cells, it was tested whether immunization with NYVAC before or after infection with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV) would increase the safety of Dryvax vaccination. NYVAC was shown to be safer in severely immunocompromised macaques and that NYVAC priming resulted in a faster resolution of Dryvax-induced lesions in both healthy and immunocompromised macaques (Edghill-Smith et al., 2003).

Rabbit Response

• Host Strain: New Zeland White (NZW)
• Vaccination Protocol: Groups of 20 NZW rabbits (10 male and 10 female) were vaccinated with LC16m8 (at approximately 2 × 10^5 PFU) via scarification to the hind flank. 28 d after vaccination, animals were challenged intradermally with either low (200 PFU) or high (1000 PFU) doses of RPV, which correspond to 1 or 5 times the LD100 value, respectively. Animals were monitored daily for temperature and behavioral changes. Survival was determined at 10 d after RPV challenge, at which time all living rabbits were euthanized (Empig et al., 2006).
• Persistence: (Empig et al., 2006)
• Side Effects: A mild inflammatory response was detected over the vaccination site 1–7 d following vaccination with either LC16m8 or Dryvax. The response was characterized by ulceration and erythema, followed by pock formation and a small scar. Despite limiting vaccination to healthy individuals in recent vaccination campaigns, adverse reactions were still observed, highlighting the need for a safer yet equally protective alternative to Dryvax. However, no serious adverse reactions, such as encephalitis, were observed during the early use of LC16m8 vaccine (Empig et al., 2006).
• Efficacy: Rabbits vaccinated with LC16m8 survived lethal RPV challenge at both challenge doses tested (Empig et al., 2006).
• Description: To evaluate the protective efficacy of LC16m8 in comparison to Dryvax, the study employed both rabbit and mouse models of poxvirus disease (Empig et al., 2006).